Anti-helicobacter activity of celery seed extract

ABSTRACT

The application discloses celery seeds or celery seed extract for treating  Helicobacter pylori  infections.

The inventor relates to the use of biologically active celery seedextracts to inhibit the growth and replication of the bacterium,Helicobacter pylori.

Arthritis and rheumatism are important world-wide problems, Around 1% ofthe UK population are affected at some stage in life. Complaints of thisnature not only cause significant disability but may also have aseverely detrimental effect on the psychological state of the sufferers.Conventionally these complaints are treated with analgesic/antipyreticdrugs and non-steroid anti-inflammatory drugs (NSAIDs) However NSAIDscan have serious side effects, such as gastotoxicity, causing forexample gatric ulceration, and hence research has been made intoalternative sources of anti-inflammatory drugs. In particular compoundsextracted from higher plants have been considered. Lewis et al (1985)and Whitehouse et al (1999) found that the extracts of celery (Apiumgraveolens) (CSE) had significant anti-inflammatory activity in animalmodels with reduced adverse effects A further risk factor in thepathogenesis of peptic ulcer disease is H. pylori infection. Chan (1997)found that eradication of H. pylori before NSAID therapy reduced therisk of ulcer development by about fourfold PCT/US99/25873 discloses theuse of celery seed extract for the prevention and treatment of paininflammation and gastrointestinal irritation.

The inventors have surprisingly found that components of celery seedextract may be used to control the growth of Helicobacter pylori.

The invention provides the use of celery seed or celery seed extract(CSE) for the inhibition of growth and replication of Helicobacterpylori

A preferred CSE is produced by supercritical fluid extraction of thestarting product, By CSE we mean a natural product derived from celeryseed, or a pharmaceutical equivalent thereof. This is preferably anethanol/water extract, especially 50% to 90%, 60% to ,5%. mostpreferably an 80% Vol:Vol ethanol/water extract. The term includes theisolated compounds obtainable from CSE.

Preferably the active component of the celery seed extract is selectedfrom the group: 3-n-butyl 4,5-dihydrolphtlide, 3-n-butyl phthalide,α-Budesmol, β-Budesmol dioctyl phthalate and cis, cis-9,12-Octadecadienoic acid.

The invention further provides a phannaceutical composition for theinhibition of growth and replication of Helicobacter pylori, comprisingcelery seed extract.

Also provided is the use of celery seed or celery seed extract in thepreparation of a pharmaceutical composition for the treatment ofHelicobacter pylori infection.

Preferably the H. pylori infection is in a al, such as a human.Preferably the infection is within the digestive tract, especially thestomach of the mammal.

The pharmaceutical composition may be administered orally, e.g. in theform of an oral suspension, solution or tablet. Dosages may be 300-2000mg. daily in divided doses preferably or even higher.

The pharmaceutical composition may comprise one or more pharmaceuticallyacceptable carriers, bulking agents or excipients known in the art (e.g.in the form of a tablet or injectable solution).

A further aspect of the invention provides celery seed or celery seedextract for use in the manufacture of a medicament to treat aHelicobacter pylori infection.

The invention will now be described in detail with reference to thefigures in which:

Table 1 shows the effect of the crude extract of CSE on the growth ofdifferent strains (3330, 3336 and 3339) of H. pylori.

Table 2 shows the distribution of antimicrobial activity against H.pylori (strain 3339) in the crude extract and different fractions ofCSE.

Table 3 shows antimicrobial activity of the subtractions from pet. etherfraction against H. pylori (strain 3339).

Table 4 shows antimicrobial activities of compounds from subfractions 6and 10 against H. pylori (strain 3339).

FIG. 1 shows the effect of CSE crude extract on the growth of thestrains (3330, 3336, 3339) of H. pylori

FIG. 2 shows the bioassay-guided frationation scheme of celery seedextract (antimicrobial agents enclosed in boxes).

FIG. 3 shows the antimicrobial activity of pet. ether fraction andsubfractions 6 and 10 against H. pylori (strain 3339).

FIG. 4 shows the analytical separation of mixture from subfraction 10.Column: Nucleosil® C18, 250×4.6 mm. I.D.; Mobilo phase: ACN/water(60:40); Flow ram: 1.0 m./min; Detection: WV @ 236 nm; Injection volume:10 jig in 1 ml of 40% ACN in water; Temperature: Ambient; ATT.3.

FIG. 5 shows the antimicrobial activities of compounds against H. pylori(strain 3339)

FIG. 6 shows the EI-MS spectrum of compound 6-1

FIG. 7 shows the ¹H NMR spectrum of compound 6-1

FIG. 8 shows the ¹³C NM spectrum of compound 6-1

FIG. 9 shows the EI-MS spectrum of compound 6-1

FIG. 10 shows the EI-MS spectrum of compound 6-3

FIG. 11 shows the EI-MS spectrum of compound 6-4

FIG. 12 shows the EI-MS spectrum of compound 10-1

Antimicrobial Test

Bacterial Strains

Three strains of H. pylori (3330, 3336 and 3339) isolated from Britishpatients with gastric ulcer (duodenal ulcer or gastritis) were studied.The identities of H. pylori were confirmed by Gram stain and ureasereaction. The bacteria were stored at −80° C. in aliquots of 1 ml ofbrocella broth containing 15% (v/v) glycerol (Kitsos and Stadtiander,1998).

Celery Seed Extract (CSE)

Test CSE was provided as dark green highly viscous liquid (supplied byBeagle International Pty. Ltd, Nerang, Qld., Australia). Initally CSEwas dissolved in dimethylsulfoxide (DMSO) as stock solution (100 mg/ml,final DMSO concentration in cultures ≦1%).

Media

For the Brucella broth (BB), (BBL, USA), Brucella (28 g) was added to ILof distilled water. After the medium was autoclaved at 120° C. for 15mins, fetal bovine serum (50 ml) was added (Morgan et al, 1987).

Inocula

Thawed isolates were inoculated onto chocolate agar plates (Mërieux) andincubated under microacroplJnllic conditions (85%N₂, 10%CO₂, 5%0₂) for48 h at 37° C. Colonies were suspended in 5ml of Brucella broth andadjusted to a turbidity equivalent to a No.2 McFarland standard(approximately 6×10⁸ CFU/ml) for broth dilution method. The finalinoculum was 10⁷ CFU/ml for agar dilution method by a further 50-folddilution.

Broth Dilution Test

The CSE suspension (1 mg/ml) was serially two-fold diluted in BB. Theconcentrations (1000, 500, 250, 125, and 62.5 μg/ml) were obtained. Thesolutions were added to the column wells of 24-well plate each in. equalvolume (1 ml/well). 20 μl of cell suspension was inoculated into eachrow wells of 24-well plates (except last row wells). The culture disheswere gently agitated following the addition of the inoculum and thenplaced at 37° C. under microaeophilic conditions for three days. At theend of incubation, 1 ml of bacterial culture solution from each wellwere diluted to one in a million dilution (10⁻⁶). Then 20 μl aliquotsfrom each solution were transferred to columbia agars and incubated foran additional three days. Generally, only spots with between 7-11colonies were counted. Growth was determined on the basis of calculatingthe number of bacteria per milliliter (numbers of ba/ml=numbers ofcolonies on plate×reciprocal of dilution of sample). Bacteria growth,culture medium and extract controls were run in parallel. (Osato et al,1999).

Chromatograplic Methods

Column chromatography was performed on silica- gel 60 (40-60 μm, Merck).Analytical thin layer chromatography (TLC) was carried out on precoatedsilica gel 60 F₂₅₄ plates (layer thickness 0.2 min, Merck), developedwith the following solvent, hexane-EtOAc (70:30), chloroform-methanol(98:2). For isolation monitoring, spots were located by their absorptionunder ultraviolet (UV) light (254 and 366 nm) directly. After that theplates were sprayed with anisaldehyde reagent and heated at 110° C. for5 min (Dey and Harbourne, 1991).

HPLC (1090 LC, Hewlett Packard, UK) Analytical and Semi-PreparativePurification

Analytical Conditions

Analytical column: Nucelosil® C18, particle size 5 μm, 250×4.6 mm I.D.,catalogue No.89141 (Alltech, Carnforth, Lancashire, UK)

Mobile phase: acetonitrile/water (60:40)

Flow rate: 1.0 m/min

Injection volume: 10 μl

Detection: UV @236 nm

Sample: mixture of compounds 10-2, 10-3 and 10-4 (Conc.=5 mg/ml)

Temperature: ambient

ATT: 3

Semi-preparative conditions:

Semi-preparative column: Luna C18(2), particle size 5 μm, 250×10.00 mmI.D., catalogue No.00G-4252-NO (Phenomenex, Macclesfield, Cheshire, UK)

Mobile phase: acetonitrile/water (60:40)

Flow rate: 5.0 ml/min

Injection volume: 100 μl

Detection: UV @ 236 nm

Sample: mixture of compounds 10-2, 10-3 and 10-4 (Cone. 5 mg/ml)

Temperature: ambient

ATT: 6

Spectroscopic Methods

Mass spectrometry (MS)

The Mass spectra were recorded on a VG 70/70 Sector Mass Spectrometerinstrument (Micromas, Manchester, UK) in the Laboratory of Biomedicalresearch center (Sheffield Hallam University).

Nuclear magnetic resonance (NMR)

NMR spectra were recorded in CDCl₃ at RT on a Bruker Unity Ac 250 MHz(¹H 250 MHz; ¹³C, 62.9 Mhz).

Results and Discussion

The 80%/ ethanol extract exhibited appreciable antimicrobial activity atthe minimum inhibitory concentrations (MIC) of 250, 125 and 125 μg/ml,respectively, against H. pylori strains 3330, 3336 and 3339. The resultsof antimicrobial activity of CSE are given in Table 1 and FIG. 1. Thebioassay-guided frationation scheme of CSE is illustrated in FIG. 2. Thefrationation for the isolation of the active compounds was performedfrom the 80% ethanol extract of CSE. The susceptibility of H. pyloristrain 3339 was higher than 3330 and 3336. Later, in antimicrobialactivity testing of fractions and subfractions of CSE, only H. pylori3339 strain was chosen for fractionation guide. The residue of 80%ethanol extract of CSE was subsequently successively partitioned withorganic solvents and water. The activity emerged predominantly in thepetroleum ether layer (MIC-15.625 μg/ml) as compared to the othersolvents, diethyl ether (MIC=125 μg/ml), ethyl acetate (MIC>500 μg/ml)and water (MIC>500 μg/ml) (Table 2).

The petroleum ether fraction was directly subjected to coltchromatography on silica gel with hexane, hexane-EtOAc (99:1),hexane-EtOAc (95:5) hexane-EtOAc (70:30) and EtOAc as eluent. Fractionswith the same retardation factors were combined to yield 11 majorfractions. Each subfraction was tested for antibacterial activityagainst H. pylori. The results of the antimicrobial testing of thedifferent subfractions are shown in Table 3. The most pronouncedantimicrobial activity successively resided in the subfraction 6 elutedwith hexane-EtOAc (95:5) (MIC=15.625 μg/ml) and the subfraction 10eluted with hexane-EtOAc (70:30) (MIC=15.625 μg/ml (FIG. 3). Subfraction6 was further purified by silica gel column chromatography(hexane-ether, 10:1, as solvent) and preparative TLC usingchloroform/pet. ether (3:1) to yield compounds 6-1, 6-2, 6-3 and 6-4.Subfraction 10 was further purified with hexane-ether (7:3) as mobilephase to afford a pure compound 10-1 and a mixture. The mixture wasdissolved in 40% ACN in water and passed through the DPA-6S SPE column(Supelco, UK) to remove the chlorophyll. The eluate with methanol wasevaporated to dryness and reconstituted in 40% ACN in water for HPLCanalysis. It was separated into three compounds 10-2, 10-3 and 10-4 byanalytical HPLC using ACN/water (60:40) as mobile phase (FIG. 4). Largequantity of individual pure compounds will be obtained bysemi-preparative HPLC and sent for MS and NMR spectroscopic analysis.

Compounds 6-1, 6-2, 6-3, 10-1 and the combination of 6-1 and 6-3 wereevaluated for antimicrobial activity. The results indicated they werepartly responsible for the antimicrobial activity of CSE (Table 4 andFIG. 5). The mixture of 6-1 and 6-3 by different combination did notexert a syngergistic effect in antimicrobial activity. The mixture ofcompounds 10-2, 10-3 and 10-4 showed an interesting antimicrobialactivity against H. pylori. Very recently, Momin and Nair (2001)isolated and characterized three bioactive compounds, sedanolide,senkyuolide-N and senkyunolide-J from CSE with the significantmosquitocidal, nematicidal and antifungal activities. Further study willconfirm with MS and NMR data if compounds 10-2, 10-3 and 10-4 arecorresponding to sedanolide, sekynuolide-N and sekyunolide-J. Theantimicrobial activity of individual compound will be tested as well.

The exact structures are confirmed by comparison of their physical andspectral data ([α], 1H and 13NMR) with data in the literature.Structural elucidation of the compounds isolated from active fractions 6and 10 are given below:

Compound 6-1 was obtained as pale yellow oil with a distinct celeryodour. The electron impact mass spectrometry (EI-MS) spectrum (FIG. 6)of the compounds showed the molecular ion peak at mass/charge ratio(m/z) 192 (composition, 22.9%), corresponding to the molecular formulaC₁₂ _(H) ₁₆0₂. Other major peaks were at m/z (composition, %) 163 (3.6),135 (5.3), 10B (21.7), 107 (100%), 85 (9.7), 79 (24.3), 77 (24.2) and 57(14.4).

The ¹H NMR spectrum (FIG. 7) displayed a doublet at 6.12 ppm (1H, J=10Hz) and a multiplet at 5.9 ppm for the vinyl protons, H-7 and H-6,respectively, as well as multiplet at 4.9 ppm for H-3. In ¹³C NMRspectrum (FIG. 8), the signals at 128.4, 116.8 and 124.5 ppm wereconsistent with disubstituted and tetrasubstituted double bands composedof C-6, C-7 and C-1a, C-3a, respectively. In addition, tetra substitutedsignals appeared for the side chain (C-1′, C-2′, C-3′, C-4′) in therange of 13.8-22.4 ppm. The signals due to C-1, CA and C-S appeared at161, 31.9 and 26,7 ppm.

On the basis of EI-MS and ¹H- and ¹³C-NMR, compound 6-1 was identifiedas 3-n-butyl 4,5-dihydrolphthalide (sedanenolide) (Bjeldanes and Kim,1977).

Experimental Data

Compound 6-1 EI-MS: m/Z 192.3 (calculated for C₁₂H₁₆O₂). ¹H NMR (CDCl₃):δ 0.9 (t, 3H, J=7.2, H-4′), 1.2-1.8 [m, 6H, H1(1′, 2′, 3′)], 2.45 (m,H-4,5), 4.9 (m, 1H, H-3), 5.9 (m, 1H, H-6), 6.2 (d, 1H, J=10, H-7); ¹³CNMR (CDCl₃): δ 13.8-22.4 (C-1′, 2′, 3′, 4′), 26.7-31.8 (C-4.5), 82.5(C-3), 116.8 (C-7), 128.3 (C-6), 124.5-135 (C-8, 9), 161.4 (C-1).

Compound 6-2 was obtained as pale yellow oil with a distinct celerycolour. The EI-MS spectrum (FIG. 9) of 6-2 showed the molecular ion peakas mass/charge ratio (m/z) 190, Corresponding to the molecular formulaC₁₂H₁₄O₂. Other major peaks were at m/z 163, 148, 144. 133 (100%), 115,105, 91 and 77.

On the basis of EI-MS and ¹H- and ¹³C-NMR, compound 6-2 was identifiedas 3-n-butyl phthalide (Zheng et al 1993).

Experimental Data

EI-MS: m/z 190.2 (calculated for C₁₂H₁₄O₂). ¹H NMR (CDCl3): δ 0.85 (t,3H J=7.1, H-4′), 1.2-2.10 (m, 6H, H-(1′ 2′, 3′), 5.42 (dd, 1H, J=7.8 and4.1 Hz, H-3), 7.39 (d. 1H, J=7.5, H4), 7.46 (t, 1H, J=7.5, H-6), 7.62(t, 1H, J=7.5 HZ, H-5), 7.83 (d, 1H, J=7.5 Hz, H-7); ¹³C NMR (CDCL₃):δ14.08 (C-4′), 22.65 (C-3′), 27.01 (C-1′), 34.62 (C-2′), 81.75 (C-3),121.68 (C-4), 125.57 (C-6), 125.96 (C-9), 128.94 (C-7), 134.20 (C-5),150.02 (C-8), 171.04 (C-1).

(Large quantity of 62 will be obtained by purification using PTLC orsemi-preparative HPLC, then ¹H NMR and ¹³C NMR will be acquired again toget clear spectra).

For compound 6-3, the EI-MS spectrum (FIG. 10) showed the molecular ionpeak at mass/charge ratio (m/z) 222, corresponding to the molecularformula C₁₅H₂₆O. Other major peaks were at m/z 204, 189, 162, 149, 135,109, 108, 95, 81, 59 and 41. On the basis of EI-MS, the compound 6-3 wasidentified as mixture of α and β-Eudesmol (El-Sayed et al. 1989).

¹H NMR and ¹³C NMR spectra will confirm the structure of 6-3. But thereis not enough sample by now for measuring ¹H NMR and ¹³NMR (around 10-20mg needed). The possible structure of compound 6-3 is as below:

Compound 6-4 was obtained as colorless oil The EI-MS spectrum of 64(FIG. 11) showed tho major peaks at m/z 279, 167, 149, 83, 71, 57 and43, On the basis of EI-MS, the Compound 64 was identified as dioctylphthalate, corresponding to tire molecular formula C₂₄H₃₈O₄ (MW=390.54 )(MS library).

¹H NMR and ¹³C NMR spectra will confirm the structure of 64. But thereis not enough sample by now for measuring ¹H NMR and ¹³C NME (around10-20 mg needed). The possible structure of compound 6-4 is as below:

Compound 10-1 was obtained as a colourless oil. The EI-MS spectrum (FIG.12) of 10-1 showed the molecular ion peak at mass/charge ration (m/z)280, corresponding to the molecular formula C₁₈H₃₂O₂. Other major peakswere at m/z 137, 123, 109, 95, 81, 67, 55, 54 and 41. On the basis ofEI-MS, the compound 10-1 was identified as linoleic acid (cis, cis -9,12- Octadecadienoic acid) (MS library).

¹H NMR and ¹³C NMR spectra will confirm the structure of 10-1. But thereis not enough sample for measuring ¹H NMR and ¹³C NMR (around 10-20 mg).

The possible structure of compound 10-1 is as below:

CONCLUSION

Overall the CSE has showm interesting antimicrobial activity against H.pylori. Five compounds have been purified which arc partly responsiblefor the antimicrobial properties. The structure elucidation of compoundsis still undergoing. Further work will continue to purify the activeconstituents in subfraction 10 and other subtractions and to test theanti-cytokine activity and cartilage protection properties. If thecompounds from subfractions 6 and 10 are not responsible for theanti-inflammator activity, the constituents maybe reside in otherfractions and subfractions.

REFERENCES

-   Bjeldanes L. F. and KIM I. S. (1977) Phthalide components of celery    essential oil. J. Org. Chem. 42(13),23333-5.-   Chan. F. K. L., Sung J. Y., Leung V. K. S. et al (1997) Randomized    trial of eradication of H. pylori before non-steroid    anti-inflammatory drug therapy to prevent peptic ulcer. Lancet 350,    975-9.-   Dey P. M. and Harborne J. B. (1991) Methods in plant Biochemistry,    volume 7, Terpenoids, Edited by Charlwood B. V. and Banthorpe D. V.,    Academic Press, p.65.-   El-Sayed A. M. Al-Yahya M. A. Hassan, M. M. (1989) Chemical    composition and antimicrobial activity of the essential oil of    Chenopodium botrys growing in Saudi Arabia. Int. J. Crude Drug Res.    27, 185-188.-   Kitsos C. M. and Stadtländer C. T., (1998) Helicobacter pylori in    liquid culture: Evaluation of growth rates and ultrastructure. Curr.    Microbiol. 37, 88-93.-   Lewis D. A., Darib S. M. and Veitch G. B. A. (1985). The    anti-inflammatory activity of celery Apium graveolens L. (Fam.    Umbelliferae) Int J. Crude Drug Res. 23, 27-32.-   Momin R. A. and Nair M. G. (2001) Mosquitocidal, and antifungal    compounds from Apium graveolens L. seeds. J. Agric. Food Chcm. 49,    142-145.-   Morgan D., Freedman R., Depew C., and Kraft W. (1987) Growth of    Campylobacter pylori in liquid media. J. Clin. Microbiol.    25,2123-2125.-   Osato M. S., Reddy S. G. and Graham, D. Y. (1999) Osmotic effect of    honey on growth and viability of H. pylori . Dig. Dis. Sci. 44,    462-464.

Zheng G. Q.; Kenney P. M.; Zhang J.; Lam L. K. T. (1993) Chemopreventionof benzo[a]pyrene-induced forestomach cancer on mice by naturalphthalides from celery seed oil. Nutr. Cancer 19(1), 77-86. TABLE 1Effect of the crude extract of CSE on the growth of different strains(3330, 3336 and 3339) on H. pylori. Strains MIC (μg/ml) MBC (μg/ml) 3330250 500 3336 125 500 3339 125 500

TABLE 2 Distribution of antimicrobial activty against H. pylori (strain3339) in the crude extract and different fractions of CSE. Fractions MIC(μg/ml) MBC (μg/ml) Crude extract 125 500 Pet. ether 15.625 31.25Diethyl ether 125 500 Ethylacetate >500 >500 Water >500 >500

TABLE 3 Antimicrobial activity of the subfractions from pet. etherfraction against H. pylori (strain 3339). Fractions and subfractions MIC(μg/ml) Pet. ether 15.625 Sub-1 >125 Sub-2 >125 Sub-3 125 Sub-4 62.5Sub-5 62.5 Sub-6 15.625 Sub-7 31.25 Sub-8 31.25 Sub-9 62.5 Sub-10 15.625Sub-11 31.25

TABLE 4 Antimicrobial activities of compounds from subfractions 6 and 10against H. Pylori (strain 3339). Compounds MIC (g/ml) MBC (g/ml)sedanenolide 31.25 62.5 3-n Butyl phthalide 15.625 N.T. Eudesmol 15.625125 Eudesmol + sedanenolide 15.625 N.T. (major) (minor) Eudesmol +sedanenolide 31.25 N.T. (minor) (major) Linoleic acid 62.5 >125 10-2,10-3 and 10-4 12.5 25N.T.: not tested

1. Use of celery seed or celery seed extract (CSE) for the inhibition ofgrowth and replication of Helicobacter pylori.
 2. Celery seed or celeryseed extract for the preparation of a pharmaceutical composition totreat Helicobacter pylori infection.
 3. Use according to claim 1 orclaim 2 wherein the celery seed extract is an ethanol/water extract. 4.Use according to any preceding claim wherein the active component of thecelery seed extract is selected from 3-n-butyl 4,5dihydrolphthalide,3-n-butyl phthalide, α-Eudesmol, β-Eudesmol dioctyl phthalate and cis,cis-9,12-Octadecadienoic acid.
 5. A method of treating Helicobacterpylori infection comprising administering a pharmaceutically effectiveamount of celery seed or a celery seed extract